In an aerobic granular sludge (AGS) reactor treating urban wastewater, nutrient removal depends on the availability of carbon source. Domestic wastewater consists of 40-60% of slowly biodegradable complex substrates, out of which proteins form a major fraction. Despite this, little is known about the mechanisms of protein degradation in AGS. This research assessed the anaerobic availability of protein substrates for enhanced biological phosphorous removal (EBPR) in an aerobic granular sludge reactor. Proteins have to be first hydrolyzed before being assimilated by the bacteria, and nutrient removal is often limited by the rate of hydrolysis. Therefore, a major part of this thesis attempted to look into the mechanisms of proteolysis in AGS. Next, the utilization of amino acids - the hydrolysis product of proteins - by PAO was explored, based on critical evaluation of available literature. Firstly, it is proposed that the important aspect likely to govern the hydrolysis of proteins in AGS is the substrate-granule interaction, taking into account the diffusion limitation of particulate substrates within the granules and the probable presence of hydrolytic enzymes on the granular surface. Further, it is seen that the amino acids may be utilized by the polyphosphate accumulating organisms (PAOs) either directly or after the anaerobic degradation of amino acids to simple VFAs (volatile fatty acids), which are then taken up by the PAOs. The anaerobic degradation or fermentation of amino acids may occur via two well-known pathways- Stickland pathway and the non-Stickland pathway. Non-Stickland reaction requires syntrophy with hydrogen consuming bacteria whose presence in AGS is questionable. The bacteria responsible for Stickland reaction are obligate anaerobes belonging to the genus Clostridium which has not been found in aerobic granular sludge. Thus, it seems more likely that the amino acids are directly taken up by the PAOs in an AGS reactor. However, the direct uptake of amino acids by the PAOs has been reported only for eleven amino acids in total. More research on the likely fate of the remaining amino acids is recommended, considering that very little is known about the fate of amino acids in AGS. Few laboratory experiments were also conducted to study the effect of substrate size and granule size on the rate of hydrolysis of proteins. From preliminary experiments, it was seen that aerobic granular sludge exhibited significant anaerobic phosphate-release activity when casein (protein) was the only available carbon source. In the lab experiment carried out with different sizes of protein substrates, the observed anaerobic phosphate-release activity was used to obtain the rate of hydrolysis of different sizes of casein. Based on the hydrolysis rate obtained, it is seen that in an AGS reactor with a typical sludge concentration of 8g/l, up to 90% of the proteins present in domestic wastewater influent could be potentially taken up by the PAOs, provided that the proteins are completely dissolved. It was also seen that 60-80% of the particulate casein COD (>0.45um) was hydrolyzed within 24 hours of the assay. In another lab experiment, fluorescent protease assay was performed to assess the effect of granule size on the rate of hydrolysis. A significant decrease (by at least 2 times) in the specific rate of protein hydrolysis was observed when the aerobic granule size was increased from 1-2mm to 3.15-4mm. This may be significant especially when the stratification of granules and plug-flow feeding in an AGS reactor is taken into account. Further research is recommended to analyze the relative effect of substrate size and granule size on the rate of hydrolysis of proteins in AGS.

The performance of AGS reactors treating municipal wastewater can be optimised by converting influent particulate matter into readily available substrate. This can be done via anaerobic hydrolysis and fermentation of the influent. Anaerobic processes taking place in pressure sewers are not fully understood but show the potential to act as a pre-treatment for the wastewater reaching AGS reactors. Moreover, the contribution of the influent to the hydrolytic activity of the reactor is unknown. This research evaluated the impact of a pressure sewer on wastewater characteristics, as a possible pre-treatment of sewage before reaching the treatment plant. The variations of sewage in terms of physicochemical composition and microbial activity were monitored in a full-scale pressure sewer, focusing on the hydrolysis and fermentation of organic matter for further treatment in AGS reactors. Moreover, the contribution of the influent to the enzymatic activity of a full-scale AGS reactor was assessed. Inaccuracies deriving from sampling on a full-scale pressure sewer might have affected the results. However, statistical analyses helped to derive trends from the collected data. The pressure sewer primarily affected the degree of fermentation of the wastewater and the concentration of suspended solids. It is hypothesised that such variations could benefit the performance of AGS reactors. Although the biodegradability and enzymatic activity of the wastewater did not improve significantly, anaerobic conveyance seemed more appropriate than aerobic transport for AGS reactors. However, the influent did not seem to have a large contribution to the total reactor activity, due to the high concentration of granular biomass.